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  • Original article
  • Open Access

Lower genomic stability of induced pluripotent stem cells reflects increased non-homologous end joining

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Contributed equally
Cancer Communications201838:49

https://doi.org/10.1186/s40880-018-0313-0

  • Received: 31 October 2017
  • Accepted: 11 June 2018
  • Published:

Abstract

Background

Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) share many common features, including similar morphology, gene expression and in vitro differentiation profiles. However, genomic stability is much lower in iPSCs than in ESCs. In the current study, we examined whether changes in DNA damage repair in iPSCs are responsible for their greater tendency towards mutagenesis.

Methods

Mouse iPSCs, ESCs and embryonic fibroblasts were exposed to ionizing radiation (4 Gy) to introduce double-strand DNA breaks. At 4 h later, fidelity of DNA damage repair was assessed using whole-genome re-sequencing. We also analyzed genomic stability in mice derived from iPSCs versus ESCs.

Results

In comparison to ESCs and embryonic fibroblasts, iPSCs had lower DNA damage repair capacity, more somatic mutations and short indels after irradiation. iPSCs showed greater non-homologous end joining DNA repair and less homologous recombination DNA repair. Mice derived from iPSCs had lower DNA damage repair capacity than ESC-derived mice as well as C57 control mice.

Conclusions

The relatively low genomic stability of iPSCs and their high rate of tumorigenesis in vivo appear to be due, at least in part, to low fidelity of DNA damage repair.

Keywords

  • Genomic stability
  • DNA damage repair
  • iPSCs
  • ESCs

Background

Embryonic stem cells (ESCs) are pluripotent and could differentiate into all types of somatic cells [1]. ESCc have enormous potential in the treatment of a variety of diseases, but their clinical application has been limited by ethical controversy. In 2006, Yamanaka and colleagues overexpressed four transcription factors (Oct4, Sox2, c-Myc and Klf4) in mouse somatic cells and obtained ESC-like pluripotent stem cells, termed induced pluripotent stem cells (iPSCs) [2]. iPSCs resemble ESCs in morphology, gene expression profile, epigenetic status and in vitro differentiation capacity. The development of iPSCs raises new hope for personalized clinical therapy [35].

The four transcription factors (Oct4, Sox2, c-Myc and Klf4) that are critical for the production of iPSCs are frequently overexpressed in various cancers, and mice derived from iPSCs are prone to develop tumors [69]. Although only a small population of transformed cells with genetic mutations is likely to develop into tumors [10], the genomic instability of iPSCs is a major concern that could produce huge impact on their eventual clinical use [1116].

One possible explanation for the observed greater genomic instability of iPSCs is alterations in the fidelity of DNA repair pathways. Double-stranded DNA breaks, for example, can be repaired via homologous recombination (HR) with high fidelity, or via non-homologous end joining (NHEJ) with lower fidelity [1720]. In the current study, we examined whether iPSCs differ from other types of pluripotent cells in their ability to perform these types of DNA repair. Briefly, ionizing radiation was used to induce double-stranded DNA breaks in the following cells: mouse iPSCs induced using lentivirus (lv-iPSCs) or chemically with CHR99021, Repsox and forskolin (ci-iPSCs) [21]; mouse ESCs; and mouse embryonic fibroblasts (MEFs) [2226].

The experiments showed that lv-iPSCs are more likely than the other cell types to harbor genomic abnormalities, likely due to lower genomic fidelity of DNA damage repair. We also found greater genomic stability in ci-iPSCs than lv-iPSCs.

Methods

Cell lines and culture

The lv- and ci-iPSCs were derived from female transgenic OG2 mice carrying an Oct4-GFP transgene. Both types of iPSCs and ESCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 15% fetal bovine serum (FBS; Gibco), 1% MEM non-essential amino acids (Gibco), 1% penicillin/streptomycin (Gibco), 2 mmol/L l-glutamine (Gibco), 1 × 103 units/mL of mouse leukemia inhibitory factor (Millipore, Temecula, CA, USA) and 0.1 mmol/L 2-mercaptoethanol (Gibco) [27]. The medium was changed daily, and cells were passaged every 2 days using 0.25% trypsin (Thermo Fisher Scientific, Beijing, China) [28]. MEFs were cultured in DMEM supplemented with 15% FBS, 1% non-essential amino acids and 1% penicillin/streptomycin [29].

Irradiation

Cells were passaged 1 day before γ-irradiation (4 Gy) with a cobalt irradiator (Thermo Fisher Scientific). After the irradiation, cells were immediately returned to the incubator, and cultured for 4 h prior to analyses as described below.

Western blotting

To test the phosphorylation level of ATM, cells were lysed in ATM lysis buffer [20 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 0.2% Tween-20, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 2 mmol/L dithiothreitol, 50 mmol/L NaF, 500 μmol/L NaVO4, 1 mmol/L phenylmethylsulfonyl fluoride, 0.1 μg/mL aprotinin and 0.1 µg/mL leupeptin], and centrifuged, as describe previously [30].

In assays of histone modification, cells were re-suspended in 1-mL triton extraction buffer (TEB) containing 0.5% Triton X-100 and 2 mmol/L PMSF, and then lysed on ice for 10 min. The lysates were centrifuged at 1500g for 10 min at 4 °C. The pellet was washed with 1.5-mL TEB, re-suspended in 0.2 mol/L HCl, and incubated at 4 °C overnight. Samples were centrifuged at 6500g for 10 min, after which 200-µL supernatant was transferred to a new tube, and neutralized with 20-µL 2 mol/L NaOH.

Samples were separated using SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Blots were incubated with a primary antibody against one of the following proteins: phospho-ATM (1:1000; R&D Systems, Minneapolis, MN, USA), β-actin (1:3000; Beyotime Biotech, Beijing, China), H3 (1:30,000; Abcam, Cambridge, MA, USA) and H3K9me3 (1:3000; Abcam). Blots were washed three times with phosphate-buffered saline (PBS), and then incubated with a horseradish peroxidase-conjugated anti-mouse secondary antibody (1:3000; Gene Tex, San Diego, CA, USA) or anti-rabbit secondary antibody (1:3000; Abcam). Protein bands of interest were visualized using an Image Quant ECL system (GE Healthcare, Piscataway, NJ, USA).

Immunofluorescence labeling of γ-H2AX foci

Cells were passaged onto slides, exposed 24 h later to 4 Gy of γ-irradiation, and incubated at 37 °C for 4 h. Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, washed again with PBS, permeabilized for 10 min using 0.05% Triton X-100 and 0.5% NP-40, and then washed three times (5 min each) in PBS. The cells were blocked for 1 h with 2% bovine serum albumin (BSA), and then incubated for 1 h at room temperature with a mouse anti-γH2AX antibody (1:500; Millipore, Temecula, CA, USA). Cells were washed three times with PBS containing 0.05% Tween 20, and then incubated with a goat anti-mouse secondary antibody (1:800; Abcam) for 1 h in the dark at room temperature. Cells were counterstained with 0.2 mg/mL 4′,6-diamidino-2-phenylindole (DAPI, 1:2000; Sigma, Shanghai, China). Confocal images were acquired and analyzed using a TCS SP5 (Leica) microscope equipped with an HCX PL 63 × 1.4 CS oil-immersion objective lens.

DNA extraction

Three types of cells (lv-iPSCs, ci-iPSCs, ESCs) were digested with 0.25% trypsin and re-suspended in gelatin-coated dishes. After incubation at 37 °C for 15 min, supernatants were transferred to 15-mL centrifuge tubes, and cells were collected by centrifugation at 500g for 5 min at room temperature. DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).

Whole-genome re-sequencing

Whole-genome DNA libraries suitable for sequencing using an Illumina sequencing platform were generated from 1-µg genomic DNA. The DNA was sheared to approximately 300–500 bp using a Covaris S220 instrument (Life Technologies, Carlsbad, CA, USA). A total of 2× 101-bp paired-end reads were produced using the HiSeq 2000 DNA Sequencer.

The sequencing data were mapped to a reference mouse genomic sequence (mm9) using the Burrows–Wheeler alignment tool algorithm [31]. Unique alignment reads were retained for later analysis. Using the untreated cells as a control, single-nucleotide variations (SNVs) were collected using the “mpileup” tool in SAMTools as well as the UnifiedGenotyper in the GATK module [32, 33]. Quality recalibration and local realignment were performed using GATK tools before variation calling was performed. The following criteria were applied for calling mutations using pairwise samples: (1) the minimum coverage of variant sites had to be greater than 20 and base quality greater than 15; (2) the frequency of mutant SNVs had to be 0 in control samples and 0.2 in irradiated samples; and (3) the variant sites had to be supported by at least two reads on the forward strand and two reads on the reverse strand.

RNA sequencing

Total RNA was extracted from each cell line using TRIzol reagent and enriched for mRNA using oligo (dT) magnetic beads. Approximately 1-µg mRNA was fragmented and electrophoresed to isolate mRNA fragments (200–250 bases). These fragments were subjected to end repair, 3′ terminal adenylation and adapter ligation, followed by cDNA synthesis. The resulting cDNAs were gel-electrophoresed to isolate 250–300 bp fragments, and were sequenced using a HiSeq 2000 system (Illumina).

Sequencing reads were aligned to a reference sequence (GRCm37/mm9) using TopHat alignment software [34, 35]. Only uniquely aligned reads were used for transcript assembly, which was performed using Cufflinks software [36]. Read counts for each gene were calculated, and the expression levels of each gene were normalized using the “fragments per kilobase of exon model per million mapped” (FPKM) algorithm. Differentially expressed genes were filtered based on false discovery rate (FDR)-adjusted P < 0.05. The profile of differentially expressed genes was visualized and analyzed using the Bioconductor function “CummeRbund” in the R program [37]. Hierarchical clustering was performed using the “heatmap” package in R.

Generation of iPSC- and ESC-derived mice

Two cell-stage ICR embryos were electrofused to produce tetraploid embryos, and 10–15 iPSCs and ESCs were subsequently injected into the reconstructed tetraploid blastocysts. Embryos were cultured for 1 day prior to transplantation into the uterus of pseudo-pregnant mice. Caesarean sections were performed at E19.5, and the pups were fostered by lactating ICR mothers [38].

Comet assay

Mice derived from iPSCs or ESCs as well as C57 mice were treated with 4 Gy ionizing radiation. At 4 h later, bone marrow cells were isolated and re-suspended using PBS and concentrated by adding 150-μL molten 0.75% low-melting-point agarose. An aliquot of concentrated cells (60 μL) was then added to molten 0.8% normal-melting-point agarose on comet slides. The slides were incubated for 1–2 h at 4 °C with pre-chilled lysis buffer, stored in the dark at 4 °C for 20 min, then incubated with pre-chilled electrophoresis buffer (0.3 mol/L NaOH containing 0.5 mol/L EDTA, pH > 13.0). Gel electrophoresis was performed at 25 V for 20 min at 4 °C. Slides were incubated at 4 °C for 15 min with neutralization buffer (0.4 mol/L Tris, pH > 7.5), washed with 100% ethanol for 3–5 min and air-dried at room temperature. Diluted ethidium bromide (EB) solution (20–30 μL) was placed onto each dried agarose circle. Slides were then read at 100 cells/sample using a fluorescence microscope equipped with CASP DNA damage analysis software.

Results

Similar gene expression profile between lv-iPSCs and ESCs

RNA-seq analysis showed that the gene expression profile of lv-iPSCs was similar to that of ESCs but not to that of MEFs (Fig. 1a), indicating iPSC pluripotency. Since genomic stability depends on DNA damage repair, we analyzed expression of the genes involved in DNA damage repair pathways. No significant differences in the expression of such genes were found between lv-iPSCs and ESCs (Fig. 1b). We further analyzed the expression of DNA repair genes that were identified during early reprogramming of iPSCs in our previous report [39] and confirmed the up-regulation of those genes at early iPSC stages (Fig. 1c). These results suggest that DNA damage repair pathways can be reprogrammed at early iPSC stages and become similar to pathways in ESCs as reprogramming continues [39].
Fig. 1
Fig. 1

Gene expression profile of ESCs, lv-iPSCs and MEFs. a Scatter plots used to identify global trends in gene expression and differences among cell lines. b Heat maps showing the expression level of DNA damage repair-associated genes in the cell lines. Blue color indicates lowest expression; fuchsia, highest. c Re-analysis of the expression of DNA damage repair-associated genes during early reprogramming

More DNA mutations in lv-iPSCs than in other cell types after ionizing irradiation

We treated mouse lv-iPSCs, ESCs and MEFs with 4 Gy ionizing radiation to induce double-strand breaks. If not repaired properly, such breaks can result in genomic abnormalities, apoptosis and senescence [23, 26, 40]. Whole-genome DNA sequencing at 4 h after irradiation revealed more SNVs in lv-iPSCs than in the other cell types (Fig. 2a, Table 1), as well as more short indels (Fig. 2a, Table 2). MEFs showed a larger variety of copy number variations (CNVs) than the other cell types (Fig. 2a).
Fig. 2
Fig. 2

Genomic variation in each cell line after ionizing irradiation. a Circos plot showing genetic alterations in lv-iPSCs, ESCs and MEFs after irradiation, based on the corresponding untreated cells as the reference. CHR, chromosome. b, c Histograms showing the numbers of (b) single-nucleotide variations (SNVs) and (c) short insertions or deletions (indels) in each type of genomic region in each cell line. d Histogram of the number of SNVs in a coding region (CDS) in each cell line

Table 1

Summary of sequencing results

Parameter

lv-iPSCs

ESCs

MEFs

IR−

IR+

IR−

IR+

IR−

IR+

Total nucleotides sequenced (Gb)

64.1

71.5

72.0

70.9

64.7

70.1

Genome coverage (fold)

20×

21×

22×

23×

20×

21×

Total number of reads

634,852,868

708,065,514

765,056,092

754,255,820

640,447,936

693,376,402

ESCs mouse embryonic stem cells, IR ionizing radiation, lv-iPSCs lentivirus induced iPS cells, MEFs mouse embryonic fibroblasts

Table 2

Summary of somatic indels in each cell line

 

lv-iPSCs

ESCs

MEFs

Somatic indels

10,127

1041

679

 CDS

7

0

3

 Intergenic

5616

594

387

 Intron

3725

370

242

 5′ UTR

596

59

31

 3′ UTR

180

18

16

 Splice site

3

0

0

CDS coding sequence, indel insertion or deletion, UTR untranslated region

A larger number of SNVs and indels occurred in coding regions, intergenic regions, introns, 5′ untranslated regions (UTRs) and 3′ UTRs of lv-iPSCs than in other cell types (Fig. 2b, c). Irradiation was associated with the appearance of many more synonymous point mutations in coding regions in lv-iPSCs (559) than in ESCs (8) or MEFs (11) (Fig. 2d, Table 3). Similarly, many more non-synonymous point mutations in coding regions were found in lv-iPSCs (307) than in ESCs (7) or MEFs (13) (Fig. 2d, Tables 3, 4, 5, 6).
Table 3

Summary of somatic mutations in each cell line

 

lv-iPSCs

ESCs

MEFs

Somatic mutation

92,027

789

2403

 CDS

867

15

24

 Intergenic

54,086

570

1526

 Intron

35,567

173

694

 5′ UTR

5128

22

106

 3′ UTR

1379

9

53

 Splice site

0

0

0

CDS

867

15

24

 Synonymous

559

8

11

 Nonsynonymous

307

7

13

 Nonsense

1

0

0

CDS coding sequence, indel insertion or deletion, UTR untranslated region

Table 4

Frequencies of coding SNVs in ESCs exposed to ionizing radiation

#

Locus

Gene

Mutation

Amino acid change

Freq. IR− (%)

Freq. IR+ (%)

1

Chr1:46244148

Dnahc7b

T−> C

V−>A

0.0

28.57

2

Chr1:172987924

Fcgr3

A−>C

S−>A

0.0

30

3

Chr2:180330052

Ogfr

A−>G

E−>G

0.0

22.06

4

Chr8:22465823

Defa-rs1

C−>G

R−>P

0.0

20

5

Chr10:80640826

Eef2

G−>A

E−>K

0.0

30.11

6

Chr15:77465439

Apol11b

C−>A

K−>N

0.0

20

7

chrX:131293286

Armcx3

G−>A

C−>Y

0.0

21.62

IR− control cells not irradiated, IR+ irradiated cells, SNV single-nucleotide variants

Table 5

Frequencies of coding SNVs in MEFs exposed to ionizing radiation

#

Locus

Gene

Mutation

Amino acid change

Freq. IR− (%)

Freq. IR+ (%)

1

Chr1:174406441

Slamf9

T−>C

M−>T

0.0

23.68

2

Chr2:10014034

Kin

G−>A

E−>K

0.0

20.00

3

Chr4:120619977

Zfp69

A−>G

S−>P

0.0

20.00

4

Chr4:146162835

Zfp600

T−>A

I−>K

0.0

20.00

5

Chr5:72655729

Atp10d

C−>G

P−>R

0.0

27.78

6

Chr7:25371714

Zfp575

G−>A

A−>V

0.0

25.00

7

Chr7:31658366

Cd22

C−>T

R−>Q

0.0

30.56

8

Chr7:48249435

4930433I11Rik

A−>T

D−>V

0.0

23.08

9

Chr7:48249440

4930433I11Rik

G−>C

A−>P

0.0

24.00

10

Chr8:93611040

Rbl2

G−>C

R−>T

0.0

20.83

11

Chr14:52076074

Vmn2r89

C−>T

A−>V

0.0

20.00

12

Chr18:67019622

Mc4r

C−>T

G−>S

0.0

22.50

13

Chr18:70668975

Poli

C−>T

G−>R

0.0

29.03

IR− control cells not irradiated, IR+ irradiated cells, SNV single-nucleotide variants

Table 6

Frequencies of coding SNVs in lv-iPSCs exposed to ionizing radiation

#

Locus

Gene

Mutation

Amino acid change

Freq. IR− (%)

Freq. IR+ (%)

1

chr1:30861639

Phf3

C−>G

E−>Q

0.00

20.93

2

chr1:60166069

Carf

C−>T

R−>W

0.00

42.86

3

chr1:92665043

Col6a3

C−>G

E−>D

0.00

22.73

4

chr1:108649819

Kdsr

G−>T

D−>E

0.00

27.59

5

chr1:152550404

Hmcn1

C−>T

V−>I

0.00

25.00

6

chr1:166275528

Nme7

G−>A

G−>S

0.00

32.26

7

chr1:171863885

1700084C01Rik

G−>A

G−>S

0.00

25.00

8

chr1:175866740

Ifi203

G−>A

T−>M

0.00

28.57

9

chr1:186630980

Mosc1

G−>C

D−>E

0.00

23.08

10

chr1:186740013

Mark1

T−>A

E−>D

0.00

32.14

11

chr2:10112008

Itih5

T−>C

S−>P

0.00

42.31

12

chr2:31655794

Abl1

A−>G

S−>G

0.00

25.00

13

chr2:31656413

Abl1

A−>C

N−>T

0.00

24.24

14

chr2:34634942

Rabepk

T−>C

K−>E

0.00

44.44

15

chr2:34858984

Hc

C−>T

S−>N

0.00

20.51

16

chr2:79182476

Cerkl

C−>T

A−>T

0.00

24.00

17

chr2:86000090

Olfr1042

T−>C

T−>A

0.00

42.86

18

chr2:86154881

Olfr1053

A−>C

I−>M

0.00

42.86

19

chr2:86828637

Olfr1101

T−>G

Q−>P

0.00

41.67

20

chr2:87149857

Olfr1118

A−>G

K−>E

0.00

40.74

21

chr2:89033480

Olfr1226

G−>A

S−>F

0.00

57.14

22

chr2:90749357

Kbtbd4

A−>G

I−>V

0.00

36.36

23

chr2:90894429

Psmc3

A−>G

T−>A

0.00

23.53

24

chr2:91757766

Ambra1

G−>A

R−>Q

0.00

60.71

25

chr2:92815310

Prdm11

G−>A

S−>L

0.00

43.33

26

chr2:119346136

Exd1

T−>A

H−>L

0.00

35.00

27

chr2:119577973

Ltk

C−>T

G−>E

0.00

34.38

28

chr2:120104674

Pla2g4d

G−>A

P−>L

0.00

20.00

29

chr2:120265164

Ganc

C−>G

I−>M

0.00

40.00

30

chr2:120357660

Zfp106

G−>T

Q−>K

0.00

42.31

31

chr2:126412071

Slc27a2

G−>T

A−>S

0.00

24.00

32

chr2:127182455

Astl

C−>T

P−>L

0.00

30.56

33

chr2:127267842

Fahd2a

C−>A

G−>W

0.00

21.74

34

chr2:146172498

Ralgapa2

C−>T

V−>I

0.00

25.00

35

chr2:150299134

Zfp345

A−>T

L−>Q

0.00

24.00

36

chr2:153757199

Bpifb3

G−>A

M−>I

0.00

33.33

37

chr2:157822874

Tti1

T−>C

K−>R

0.00

20.59

38

chr2:157832871

Tti1

C−>T

S−>N

0.00

21.74

39

chr2:165177990

Zfp663

C−>T

R−>Q

0.00

21.74

40

chr2:165880571

Ncoa3

G−>A

S−>N

0.00

43.48

41

chr2:174471852

Zfp831

T−>C

S−>P

0.00

25.93

42

chr3:19570978

Trim55

G−>A

G−>S

0.00

29.17

43

chr3:20127155

Cpa3

T−>A

K−>I

0.00

36.36

44

chr3:65861245

Veph1

A−>G

S−>P

0.00

25.00

45

chr3:88240586

Sema4a

G−>A

A−>V

0.00

29.41

46

chr3:94167523

C2cd4d

G−>C

R−>P

0.00

22.22

47

chr3:96096266

Fcgr1

G−>A

P−>S

0.00

20.00

48

chr3:97414088

Chd1l

T−>C

S−>G

0.00

42.86

49

chr3:105789443

Ovgp1

C−>T

T−>I

0.00

21.74

50

chr3:116192199

Rtcd1

C−>T

V−>I

0.00

27.27

51

chr3:118377426

Dpyd

G−>A

S−>N

0.00

41.67

52

chr3:137770265

Mttp

T−>A

T−>S

0.00

26.09

53

chr3:142271248

Gbp1

G−>A

E−>K

0.00

30.95

54

chr4:57660898

Palm2

G−>A

V−>I

0.00

40.00

55

chr4:106415886

Fam151a

G−>A

R−>Q

0.00

51.85

56

chr4:116265516

Gpbp1l1

T−>A

S−>T

0.00

25.00

57

chr4:118154980

Tie1

T−>C

D−>G

0.00

30.30

58

chr4:119804939

Hivep3

T−>C

L−>P

0.00

25.00

59

chr4:120620061

Zfp69

T−>C

T−>A

0.00

40.00

60

chr4:120620067

Zfp69

T−>G

T−>P

0.00

37.04

61

chr4:136193988

Lactbl1

A−>G

S−>G

0.00

33.33

62

chr4:141674086

Kazn

C−>T

A−>T

0.00

20.00

63

chr4:147839151

Mtor

C−>T

R−>C

0.00

25.71

64

chr5:23825901

Kcnh2

T−>G

T−>P

0.00

34.78

65

chr5:23905831

Abcb8

T−>C

W−>R

0.00

20.00

66

chr5:64289838

0610040J01Rik

T−>A

L−>Q

0.00

24.00

67

chr5:90672580

Ankrd17

A−>G

*−>Q

0.00

28.89

68

chr5:109231028

Vmn2r8

T−>C

E−>G

0.00

20.83

69

chr5:122789758

Rad9b

A−>G

L−>S

0.00

37.93

70

chr5:138473740

Smok3a

A−>G

Q−>R

0.00

32.00

71

chr5:142948192

C330006K01Rik

G−>A

G−>R

0.00

22.86

72

chr5:146996767

1700001J03Rik

C−>T

R−>H

0.00

30.56

73

chr6:67242225

Il12rb2

T−>C

Y−>C

0.00

21.21

74

chr6:67423944

Il23r

T−>C

T−>A

0.00

26.09

75

chr6:72529697

Elmod3

T−>C

H−>R

0.00

25.00

76

chr6:123355291

Vmn2r20

G−>A

A−>V

0.00

24.00

77

chr6:124820464

Cd4

G−>A

P−>S

0.00

30.77

78

chr6:128334974

4933413G19Rik

G−>A

G−>R

0.00

23.53

79

chr6:129369539

Clec9a

A−>G

N−>S

0.00

40.91

80

chr6:132907129

Tas2r131

C−>T

R−>Q

0.00

26.09

81

chr6:141942744

Gm6614

C−>T

D−>N

0.00

32.14

82

chr6:142186044

Slco1a5

G−>A

S−>L

0.00

25.00

83

chr6:142186083

Slco1a5

G−>T

P−>H

0.00

27.27

84

chr6:142201619

Slco1a5

T−>G

D−>A

0.00

35.29

85

chr6:142251831

Iapp

G−>C

S−>T

0.00

26.92

86

chr7:3794286

Pira2

A−>G

S−>P

0.00

26.09

87

chr7:7278011

Vmn2r30

T−>A

N−>I

0.00

22.22

88

chr7:10859910

Vmn1r66

G−>A

H−>Y

0.00

38.10

89

chr7:11333654

Vmn1r71

G−>A

T−>I

0.00

50.00

90

chr7:12738597

Vmn1r78

T−>C

F−>S

0.00

20.00

91

chr7:17743709

Ceacam3

C−>G

L−>V

0.00

20.00

92

chr7:17743712

Ceacam3

A−>C

I−>L

0.00

22.50

93

chr7:18337478

Ceacam5

G−>A

R−>Q

0.00

22.73

94

chr7:18662759

Ceacam12

G−>C

G−>A

0.00

26.09

95

chr7:19672969

Dmpk

G−>A

A−>T

0.00

33.33

96

chr7:26134047

Megf8

C−>T

H−>Y

0.00

20.45

97

chr7:26261696

Ceacam1

C−>G

A−>P

0.00

20.00

98

chr7:29779122

Map4k1

T−>C

C−>R

0.00

21.05

99

chr7:31370138

Wbp7

C−>A

A−>S

0.00

31.82

100

chr7:31374957

Zbtb32

C−>T

A−>T

0.00

30.77

101

chr7:31391976

Upk1a

G−>A

T−>I

0.00

31.03

102

chr7:31696435

Mag

C−>T

V−>I

0.00

40.00

103

chr7:48299575

Gm4884

G−>T

A−>S

0.00

21.28

104

chr7:48299666

Gm4884

A−>C

H−>P

0.00

22.22

105

chr7:51608348

Shank1

G−>A

G−>S

0.00

20.00

106

chr7:54720024

Mrgpra2b

T−>A

H−>L

0.00

42.42

107

chr7:55424237

Mrgprb5

A−>G

I−>T

0.00

30.43

108

chr7:55424238

Mrgprb5

T−>A

I−>F

0.00

29.17

109

chr7:86855301

Kif7

C−>T

R−>H

0.00

42.42

110

chr7:89455314

Sh3gl3

T−>G

S−>A

0.00

40.00

111

chr7:108978612

Inppl1

G−>A

H−>Y

0.00

35.29

112

chr7:109584206

Stim1

T−>A

L−>H

0.00

24.14

113

chr7:109762976

Olfr553

G−>T

L−>M

0.00

23.26

114

chr7:109832588

Trim68

T−>C

I−>V

0.00

32.00

115

chr7:109862636

Olfr33

A−>G

F−>S

0.00

27.78

116

chr7:109872941

Olfr559

A−>T

I−>N

0.00

23.53

117

chr7:110121916

Olfr577

C−>T

A−>T

0.00

34.48

118

chr7:110234973

Olfr584

T−>C

F−>L

0.00

22.58

119

chr7:110336027

Olfr592

A−>G

H−>R

0.00

33.33

120

chr7:110399181

Dub2a

C−>G

E−>Q

0.00

42.31

121

chr7:110566860

Usp17l5

C−>A

P−>T

0.00

25.00

122

chr7:111161069

Olfr639

A−>G

I−>T

0.00

25.71

123

chr7:111161070

Olfr639

T−>C

I−>V

0.00

27.78

124

chr7:111297423

Ubqlnl

C−>G

Q−>H

0.00

23.33

125

chr7:111298754

Ubqlnl

T−>G

T−>P

0.00

31.58

126

chr7:111302579

E030002O03Rik

A−>G

V−>A

0.00

20.00

127

chr7:111560797

Trim30a

G−>C

T−>S

0.00

33.33

128

chr7:111793632

Olfr658

T−>C

T−>A

0.00

25.00

129

chr7:112009277

Dub1

C−>T

R−>C

0.00

35.00

130

chr7:112041695

Olfr666

G−>A

A−>V

0.00

29.03

131

chr7:112123974

Olfr671

C−>A

S−>I

0.00

20.00

132

chr7:112462829

Olfr689

G−>T

A−>S

0.00

21.95

133

chr7:112708033

Apbb1

G−>A

S−>F

0.00

26.32

134

chr7:114029870

Olfr706

G−>A

L−>F

0.00

23.08

135

chr7:114218082

Olfr714

G−>A

V−>I

0.00

21.43

136

chr7:115302565

Olfr485

C−>T

G−>E

0.00

28.00

137

chr7:115399125

Olfr488

T−>C

K−>E

0.00

25.00

138

chr7:115968770

Olfr514

T−>C

T−>A

0.00

25.00

139

chr7:116859980

BC051019

G−>A

T−>I

0.00

26.09

140

chr7:135022359

Zfp646

C−>G

L−>V

0.00

23.68

141

chr7:135024297

Zfp646

G−>A

E−>K

0.00

20.83

142

chr7:135026037

Zfp646

A−>G

S−>G

0.00

28.89

143

chr8:4213992

BC068157

G−>A

P−>L

0.00

52.38

144

chr8:80770162

Ttc29

C−>T

P−>L

0.00

22.86

145

chr8:86691455

4930432K21Rik

C−>A

P−>T

0.00

37.50

146

chr8:112256157

Atxn1l

C−>G

V−>L

0.00

21.88

147

chr9:21085574

Kri1

T−>C

K−>E

0.00

35.71

148

chr9:21733911

Ccdc159

G−>T

S−>I

0.00

51.72

149

chr9:22004013

Zfp872

C−>T

L−>F

0.00

34.88

150

chr9:22005064

Zfp872

G−>A

G−>E

0.00

34.15

151

chr9:22005066

Zfp872

T−>C

*−>R

0.00

32.50

152

chr9:22058381

Zfp599

C−>T

M−>I

0.00

34.62

153

chr9:35646988

9230110F15Rik

A−>G

V−>A

0.00

22.22

154

chr9:36671150

Fez1

A−>C

E−>D

0.00

34.62

155

chr9:37869528

Olfr885

G−>A

V−>M

0.00

27.27

156

chr9:41932183

Sorl1

C−>T

S−>N

0.00

32.35

157

chr9:44073278

Nlrx1

A−>C

F−>V

0.00

25.00

158

chr9:45557815

Dscaml1

G−>C

K−>N

0.00

42.86

159

chr9:50490277

Dixdc1

C−>T

R−>Q

0.00

21.74

160

chr9:55821819

Rfpl3s

G−>A

T−>M

0.00

29.03

161

chr9:58347114

6030419C18Rik

G−>A

A−>T

0.00

24.14

162

chr9:120873710

Ulk4

T−>C

I−>V

0.00

23.68

163

chr10:18244674

Nhsl1

G−>C

C−>S

0.00

21.88

164

chr10:18722769

Tnfaip3

A−>G

L−>P

0.00

28.00

165

chr10:51201543

Gp49a

C−>T

P−>S

0.00

22.22

166

chr10:51201551

Gp49a

T−>A

H−>Q

0.00

20.00

167

chr10:51203657

Gp49a

T−>C

Y−>H

0.00

27.50

168

chr10:51203677

Gp49a

T−>G

N−>K

0.00

44.74

169

chr10:53257912

Mcm9

A−>T

S−>T

0.00

37.50

170

chr10:61892173

Supv3l1

C−>T

D−>N

0.00

31.43

171

chr10:62301871

Tet1

T−>C

E−>G

0.00

27.59

172

chr10:62534718

Pbld1

G−>T

G−>V

0.00

26.67

173

chr10:62534721

Pbld1

G−>A

G−>E

0.00

26.67

174

chr10:69997479

Fam13c

T−>G

S−>A

0.00

23.68

175

chr10:82654374

Chst11

G−>A

G−>S

0.00

23.08

176

chr10:85391311

Ascl4

G−>C

G−>R

0.00

28.57

177

chr10:99909744

Tmtc3

C−>T

R−>K

0.00

30.00

178

chr10:99914062

Tmtc3

C−>T

R−>K

0.00

44.83

179

chr10:100031465

Cep290

A−>C

M−>L

0.00

47.62

180

chr10:128448679

Olfr763

T−>A

C−>S

0.00

20.00

181

chr11:5587351

Ankrd36

G−>A

V−>I

0.00

33.33

182

chr11:5587391

Ankrd36

A−>T

K−>I

0.00

22.22

183

chr11:6501551

Nacad

G−>A

P−>S

0.00

39.39

184

chr11:23264045

Usp34

A−>T

E−>D

0.00

28.57

185

chr11:29429943

Mtif2

A−>G

Q−>R

0.00

25.93

186

chr11:29607190

Rtn4

G−>C

S−>T

0.00

25.00

187

chr11:29607841

Rtn4

C−>T

A−>V

0.00

31.25

188

chr11:29646793

Eml6

G−>C

L−>V

0.00

37.14

189

chr11:32184064

Hba-a1

G−>C

G−>A

0.00

28.00

190

chr11:35622812

Rars

G−>T

A−>E

0.00

22.22

191

chr11:48988354

Btnl9

T−>C

Q−>R

0.00

39.13

192

chr11:52216575

9530068E07Rik

C−>T

A−>V

0.00

34.38

193

chr11:62078872

Adora2b

G−>A

R−>H

0.00

25.71

194

chr11:67688822

Usp43

T−>C

M−>V

0.00

24.00

195

chr11:69010998

Alox8

A−>G

V−>A

0.00

21.43

196

chr11:70584746

Zfp3

C−>A

P−>T

0.00

20.83

197

chr11:70995613

Nlrp1b

A−>T

F−>Y

0.00

21.74

198

chr11:70995614

Nlrp1b

A−>G

F−>L

0.00

23.81

199

chr11:70995616

Nlrp1b

A−>C

I−>R

0.00

22.73

200

chr11:72984698

P2rx5

C−>T

A−>V

0.00

25.00

201

chr11:96214596

Hoxb2

G−>A

E−>K

0.00

61.90

202

chr11:96772447

Cdk5rap3

C−>T

V−>I

0.00

47.50

203

chr11:101045277

Cntnap1

C−>T

S−>L

0.00

31.25

204

chr11:102935952

Plcd3

A−>T

D−>E

0.00

23.81

205

chr11:106174196

Cd79b

T−>C

M−>V

0.00

22.22

206

chr11:120146302

Bahcc1

C−>G

T−>S

0.00

21.43

207

chr12:18521595

5730507C01Rik

A−>T

N−>Y

0.00

20.00

208

chr12:21271015

Asap2

C−>T

T−>I

0.00

21.43

209

chr12:21379212

Adam17

A−>C

S−>A

0.00

20.69

210

chr12:25723341

Kidins220

G−>A

G−>S

0.00

31.82

211

chr12:32005994

Lamb1

T−>C

V−>A

0.00

25.00

212

chr12:65573550

Fscb

A−>G

S−>P

0.00

25.00

213

chr12:77031626

Syne2

G−>A

R−>K

0.00

20.00

214

chr12:77088037

Syne2

A−>G

H−>R

0.00

37.93

215

chr12:77701313

Spnb1

G−>C

D−>E

0.00

27.08

216

chr12:77713010

Spnb1

A−>T

M−>K

0.00

26.47

217

chr12:80369378

Zfyve26

T−>C

Q−>R

0.00

28.00

218

chr12:85333734

Acot2

A−>G

T−>A

0.00

22.86

219

chr12:88947186

Oog1

G−>A

E−>K

0.00

20.45

220

chr12:111906810

1700001K19Rik

T−>G

Q−>P

0.00

29.82

221

chr13:6564252

Pitrm1

C−>A

T−>K

0.00

32.14

222

chr13:6604968

Pfkp

C−>T

V−>M

0.00

34.48

223

chr13:8886000

Idi1

T−>C

S−>P

0.00

22.58

224

chr13:8958551

Idi2

A−>G

E−>G

0.00

27.27

225

chr13:9150373

Larp4b

T−>C

L−>S

0.00

34.48

226

chr13:9688439

Zmynd11

C−>T

S−>N

0.00

33.33

227

chr13:14097474

Tbce

G−>A

A−>V

0.00

22.73

228

chr13:23126330

Vmn1r214

G−>A

E−>K

0.00

42.86

229

chr13:23126981

Vmn1r214

C−>A

Q−>K

0.00

40.91

230

chr13:23309404

Vmn1r221

C−>A

L−>I

0.00

30.77

231

chr13:23309956

Vmn1r221

C−>G

L−>V

0.00

25.93

232

chr13:23579753

Btn2a2

T−>A

I−>L

0.00

34.62

233

chr13:23647236

Hist1h1d

C−>T

T−>I

0.00

37.50

234

chr13:23855668

Hist1h1a

C−>T

A−>V

0.00

29.63

235

chr13:25085054

Mrs2

T−>C

T−>A

0.00

21.74

236

chr13:40238189

Ofcc1

G−>A

P−>S

0.00

20.83

237

chr13:58445640

Kif27

T−>A

I−>L

0.00

23.81

238

chr13:70874611

Adamts16

C−>T

G−>S

0.00

24.14

239

chr13:70877487

Adamts16

G−>C

Q−>E

0.00

20.59

240

chr13:81583863

Gpr98

C−>T

E−>K

0.00

34.48

241

chr13:96284081

F2rl1

C−>T

V−>I

0.00

25.00

242

chr13:98737291

Rgnef

G−>C

A−>G

0.00

33.33

243

chr14:45342600

Gm8267

A−>T

M−>K

0.00

28.00

244

chr14:50393514

3632451O06Rik

A−>G

V−>A

0.00

24.44

245

chr14:51135131

Olfr742

A−>G

N−>S

0.00

25.81

246

chr14:55283622

Acin1

T−>C

K−>E

0.00

43.18

247

chr14:70175997

Tnfrsf10b

C−>G

P−>A

0.00

27.78

248

chr14:70176001

Tnfrsf10b

T−>C

V−>A

0.00

25.00

249

chr14:78484964

AU021034

A−>G

C−>R

0.00

22.22

250

chr15:41697429

Abra

G−>C

L−>V

0.00

24.00

251

chr15:41701040

Abra

G−>C

L−>V

0.00

27.27

252

chr15:54965030

Deptor

A−>T

M−>L

0.00

25.00

253

chr15:66523859

Tg

G−>A

V−>I

0.00

20.00

254

chr15:75937421

Eppk1

C−>T

V−>I

0.00

25.00

255

chr15:76539950

Recql4

A−>G

L−>P

0.00

20.00

256

chr15:95455328

Dbx2

C−>T

V−>M

0.00

25.00

257

chr16:32756226

Muc4

C−>G

Q−>E

0.00

24.32

258

chr16:32779211

Muc4

C−>A

Q−>K

0.00

23.33

259

chr16:45577986

Slc9a10

C−>T

A−>V

0.00

20.00

260

chr16:56668453

Abi3bp

C−>A

P−>Q

0.00

22.22

261

chr16:58872574

Olfr176

C−>G

S−>T

0.00

20.59

262

chr17:6009735

Synj2

T−>A

F−>L

0.00

25.81

263

chr17:6037828

Synj2

C−>G

H−>D

0.00

25.00

264

chr17:7530924

Tcp10a

C−>T

P−>S

0.00

29.03

265

chr17:24111200

Prss30

A−>C

D−>E

0.00

21.05

266

chr17:24583771

E4f1

C−>T

S−>N

0.00

27.27

267

chr17:28021909

Uhrf1bp1

G−>A

G−>D

0.00

22.50

268

chr17:31365125

Ubash3a

C−>T

P−>S

0.00

25.81

269

chr17:31392140

Rsph1

G−>T

P−>Q

0.00

25.71

270

chr17:31398701

Rsph1

G−>A

T−>M

0.00

30.30

271

chr17:31754132

Cbs

T−>C

D−>G

0.00

29.03

272

chr17:32758655

Gm9705

G−>A

V−>M

0.00

20.00

273

chr17:33158753

Zfp763

C−>T

A−>T

0.00

22.86

274

chr17:33472542

Zfp81

A−>G

M−>T

0.00

27.59

275

chr17:34087468

B3galt4

T−>C

N−>S

0.00

28.21

276

chr17:34338203

Psmb8

G−>T

A−>S

0.00

28.57

277

chr17:34870392

C4b

C−>T

R−>Q

0.00

22.58

278

chr17:34974426

Dom3z

T−>C

L−>S

0.00

50.00

279

chr17:35267082

Apom

G−>T

Q−>K

0.00

43.33

280

chr17:35457904

H2-Q1

C−>T

P−>L

0.00

32.50

281

chr17:36168621

H2-T23

C−>G

R−>T

0.00

28.57

282

chr17:36218438

H2-Bl

T−>C

H−>R

0.00

27.03

283

chr17:36254622

H2-T10

G−>A

P−>S

0.00

33.33

284

chr17:36323554

H2-T3

T−>G

M−>L

0.00

21.67

285

chr17:43615815

Mep1a

T−>C

T−>A

0.00

25.00

286

chr17:43615911

Mep1a

T−>C

T−>A

0.00

30.00

287

chr17:43822205

Cyp39a1

G−>A

G−>R

0.00

28.57

288

chr17:46161537

Vegfa

G−>A

P−>L

0.00

37.04

289

chr17:46550212

Zfp318

A−>G

E−>G

0.00

29.03

290

chr17:46635998

BC048355

A−>C

K−>N

0.00

31.82

291

chr17:46893214

Ptcra

G−>T

R−>S

0.00

37.14

292

chr17:72047254

Fam179a

T−>G

F−>C

0.00

37.50

293

chr17:80734673

Arhgef33

G−>A

A−>T

0.00

26.67

294

chr17:88958139

Klraq1

T−>C

M−>T

0.00

29.03

295

chr17:89110812

Gtf2a1l

G−>A

R−>Q

0.00

31.03

296

chr17:89153211

Lhcgr

T−>C

T−>A

0.00

57.58

297

chr18:37907724

Pcdhga10

C−>A

H−>N

0.00

26.09

298

chr18:38132920

Arap3

G−>A

A−>V

0.00

44.83

299

chr18:60977711

Tcof1

C−>A

A−>S

0.00

56.00

300

chr18:60992401

Tcof1

C−>A

A−>S

0.00

46.43

301

chr18:65901869

5330437I02Rik

T−>C

F−>L

0.00

20.59

302

chr18:80326155

Adnp2

A−>G

F−>L

0.00

26.83

303

chr18:80389581

Rbfa

C−>T

A−>T

0.00

32.35

304

chr19:10751147

Pga5

C−>G

V−>L

0.00

45.83

305

chr19:11038598

Ms4a10

C−>T

V−>I

0.00

20.69

306

chr19:18912582

Trpm6

A−>G

M−>V

0.00

21.88

307

chr19:25696788

Dmrt3

C−>T

T−>M

0.00

29.63

308

chr7:111207208

Olfr643

G−>A

R−>*

0.00

29.41

IR− control cells not irradiated, IR+ irradiated cells, SNV single-nucleotide variants

Similar gene expression profile in lv-iPSCs with or without ionizing radiation

To determine whether ionizing radiation alters the expression of certain genes in lv-iPSCs that may help explain the high mutation rate, RNA-seq analysis was conducted in irradiated versus control cells. The results indicated a similar gene expression profile with or without radiation (Fig. 3a). In fact, irradiation appeared to up-regulate only 46 genes in ESCs and 30 genes in lv-iPSCs (Fig. 3b). In contrast to the genes in lv-iPSCs that radiation up-regulated, majority of the genes up-regulated in ESCs is implicated in cellular response to stress and cell cycle processes (Fig. 3c, d).
Fig. 3
Fig. 3

Gene expression levels in cells exposed or not to ionizing radiation (IR) for the indicated periods. a Heatmap showing Pearson’s correlation coefficients relating expression levels between irradiated and non-irradiated cells. b Volcano plots of genes expressed in irradiated and non-irradiated cells, showing genes significantly up-regulated (red dots) or down-regulated (green dots) in irradiated cells. Differentially expressed genes were filtered based on FDR < 0.05. c, d Histograms of gene ontology classifications of differentially expressed genes in irradiated (c) ESCs and (d) iPSCs. e Heat maps showing the expression level of DNA damage repair-associated genes in irradiated (+) and non-irradiated (−) cells. Blue indicates lowest expression; fuchsia, highest. BER base excision repair, HR, homologous recombination, MMR mismatch repair, NER nucleotide excision repair, NHEJ non-homologous end joining

Expression levels of genes involved in DNA damage repair pathways were higher in lv-iPSCs and ESCs than in MEFs, and ionizing radiation did not substantially alter the expression of these genes (Fig. 3e). Thus the genomic instability of lv-iPSCs is unlikely to reflect changes in the expression level of genes involved in DNA damage repair.

Weaker DNA damage repair response to ionizing radiation in lv-iPSCs

The phosphorylated histone variant H2AX (γ-H2AX) is a marker of double-strand breaks. Ionizing radiation significantly increased the number of γ-H2AX foci in lv-iPSCs, ESCs and MEFs, but the magnitude of decrease was much smaller in lv-iPSCs (Fig. 4a), suggesting lower capacity to repair DNA damage.
Fig. 4
Fig. 4

The phosphorylation level of DNA repair-associated proteins. a Quantification of the numbers of γ-H2AX foci in lv-iPSCs, ESCs and MEFs. Error bars represent the standard error of the mean (SEM) for the numbers of γ-H2AX foci per nucleus based on 4–5 fields, each containing approximately 20–30 cells. Significance of differences was assessed using Student’s t test. **P < 0.01 (three independent experiments). b Western blot analysis of phosphorylated ATM (p-ATM) and phosphorylated catalytic subunit of DNA protein kinase (p-DNA-PKcs) in lv-iPSCs and ESCs before and after ionizing irradiation. c Western blot analysis of the trimethylation level of H3K9 in lv-iPSCs and ESCs before and after ionizing irradiation

Next we tested whether the lower genomic stability of lv-iPSCs reflects deficiency in the error-free HR repair pathway. Indeed, we found ATM phosphorylation to be defective in lv-iPSCs (Fig. 4b) [30, 41]. We also found lower levels of H3K9me3, which recruits repair proteins to double-strand breaks, in irradiated lv-iPSCs than in irradiated ESCs or MEFs (Fig. 4c). All together, these findings may help explain the higher mutation rate of lv-iPSCs.

Lower genomic stability in lv-iPSCs than ci-iPSCs

Treatment with ionizing radiation led to higher levels of phosphorylated ATM in ci-iPSCs than in lv-iPSCs (Fig. 5a). This may help explain the higher genomic stability of ci-iPSCs [41]. Whole-genome re-sequencing at 4 h after irradiation revealed 1709 SNVs in the ci-iPSCs; this was slightly more than in treated ESCs but far less than in lv-iPSCs (Fig. 5b). Similarly, the proportion of SNVs in coding sequences, introns, 5′ or 3′ UTRs and intergenic regions was slightly higher in ci-iPSCs than in ESCs, but much higher in lv-iPSCs (Fig. 5c, d). These results indicate greater genomic stability in ci-iPSCs than in lv-iPSCs, which is due at least in part to greater activity of the HR pathway of DNA damage repair.
Fig. 5
Fig. 5

High genome stability of ci-iPSCs. a Western blot analysis of phosphorylated ATM (p-ATM) in ci-iPS, lv-iPS and ESCs before and after ionizing irradiation. b Circos plot showing genetic alterations in irradiated ci-iPSCs and ESCs, based on the corresponding untreated cells as a reference. Chromosome numbers are indicated as the outermost labels. c Histograms showing the number of SNVs in each genomic region of irradiated lv-iPSCs, ci-iPSCs and MEFs. CDS coding sequence, SNV single-nucleotide variants, UTR untranslated region. d Histograms showing the numbers of SNVs in the coding regions of irradiated lv-iPSCs, ci-iPSCs and MEFs

lv-iPSCs can tolerate more genomic DNA variation

The abovementioned results led us to hypothesize that lv-iPSCs can survive with greater genomic variation than the other cell types. Consistent with this hypothesis, we found that lv-iPSCs indeed had more DNA variation than the other cell types, yet the percentage of apoptotic lv-iPSCs did not increase between 24 and 48 h after irradiation (Fig. 6a) and the rate of lv-iPSC proliferation was greater than that of ESCs or MEFs (Fig. 6b). When we analyzed whether irradiation arrested lv-iPSCs in the G2/M phase, we observed a high proportion of arrested cells at 24 h after irradiation, but a lower proportion at 48 h (Fig. 6c). We observed similar results with ESCs, showing an increased proportion of ESCs in G2/M phase at 24 h after irradiation and a lower radiation arrest at 48 h. These results suggest that lv-iPSCs tolerate greater genomic DNA variation than the other cell types.
Fig. 6
Fig. 6

Greater tolerance of genomic DNA variation in lv-iPSCs. a Flow cytometric analysis of apoptosis rate in lv-iPSCs, ESCs and MEFs following ionizing irradiation (IR) for the indicated periods. 7-AAD 7-amino-actinomycin. b Cell proliferation rate (based on BrdU incorporation) in lv-iPSCs, ESCs and MEFs exposed to ionizing irradiation for the indicated periods. Each point represents a mean of three replicates **P < 0.01. c Analysis of cell cycle distribution in lv-iPS, ESCs and MEFs exposed to ionizing radiation for the indicated periods

lv-iPSCs are more susceptible to DNA damage

Next we compared genomic stability in mice derived from lv-iPSCs versus ESCs. C57 mice were included as additional control. Irradiation of the mice led to a higher percentage of impaired bone marrow cells (Fig. 7a–c) and of tail DNA in bone marrow cells (Fig. 7d) in iPSC-derived mice than in ESC-derived mice and C57 mice. These results suggest that mice derived from lv-iPSCs have lower DNA damage repair capability than ESC-derived or C57 mice and are therefore more susceptible to DNA damage.
Fig. 7
Fig. 7

Genome stability of mice derived from lv-iPSCs or ESCs following exposure to ionizing radiation (IR). Controls were C57 mice. a Mice were generated from lv-iPSCs or ESCs through tetraploid embryo complementation. Representative results from three independent experiments are shown. b Examples of bones from the three types of mice, from which marrow cells were extracted. c Box plots showing the percentage of impaired bone marrow cells in each mouse strain. DNA damage was evaluated using single-cell gel electrophoresis **P < 0.01. d Box plots showing the percentage of Tail DNA in impaired cells as a measure of DNA damage. Tail DNA% = Tail DNA intensity/Cell DNA Intensity × 100%. CASP software was used to calculate tail moment based on 50–100 randomly selected cells per sample

Taken together, our in vitro and in vivo experiments suggest that lv-iPSCs are more sensitive to environmental stress than ci-iPSCs, ESCs or MEFs. Ionizing radiation induces higher genomic mutation rates in lv-iPSCs, which nevertheless better tolerate the resulting genomic alterations. Genomic mutations that accumulate in lv-iPSCs are passed onto the next generation, resulting in genomic instability (Fig. 8).
Fig. 8
Fig. 8

Diagram illustrating factors influencing the genome stability of iPSCs. Environmental factors contribute to genomic variations in lv-iPSCs. In response to double-strand DNA breaks, lv-iPSCs always adopt the error-prone NHEJ repair pathway. The resulting low fidelity of DNA repair makes the lv-iPSC genome unstable and the cells more vulnerable to environmental stress. Genomic stability of iPSCs appears to depend on the method used to generate them: ci-iPSCs show greater stability than lv-iPSCs. HR homologous recombination repair pathway, IR ionizing radiation, NHEJ non-homologous end joining

Discussion

Reprogramming to generate iPSCs more efficiently [29, 4251] has been linked to the accumulation of genomic abnormalities [5259]. This poses a problem for the use of iPSCs, since mice derived from such cells can tolerate the accumulation of somatic mutations for up to six generations [60]. In the present study, we used whole-genome sequencing to compare the genomic stability of iPSCs prepared using lentivirus or chemically, and to benchmark that stability against ESCs and MEFs. We found that ionizing irradiation led to the highest rate of somatic mutations and short indels in lv-iPSCs, and this correlated with low levels of ATM phosphorylation, indicating low fidelity of DNA damage repair [41]. Experiments in vitro and in mice derived from lv-iPSCs showed that this type of pluripotent cell tolerates genomic mutations better than the other cell types evaluated.

Although iPSCs resemble ESCs in morphology, gene expression profile and in vitro differentiation capacity, they differ substantially in genomic stability. The low fidelity of DNA repair observed in our study suggests that irradiation of lv-iPSCs induces a high rate of genomic abnormalities, which is less likely to trigger apoptosis in these cells and is therefore more likely to be tolerated, thus leading to a high rate of tumorigenesis in vivo. Compromised error-free HR pathway of DNA damage repair in lv-iPSCs may help explain the relatively high genomic instability in these cells. Indeed, inhibiting the HR pathway in iPSCs has been shown to destabilize the genome [61].

Our results suggest that the epigenetic status of iPSCs may contribute to, or modulate, their genomic instability. Variation in levels of H3K9me3 and phosphorylated ATM among iPSCs may mean that cells vary in their reliance on DNA damage repair pathways, which vary in their fidelity. Future studies should further examine the potential involvement of epigenetics and other factors in iPSC genomic instability.

Future work is also needed to clarify to what extent factors that are intrinsic or extrinsic to stem cells determine the risk of malignant transformation. Tomasetti et al. found that cancer risk in certain tissues correlated strongly with the number of divisions that the stem cells had undergone, suggesting that the accumulation of genomic mutations is primarily responsible for high risk of tumorigenesis [62]. Another study, in contrast, suggested that intrinsic factors account for only 10%–30% of cancer risk, with the majority of the risk due to extrinsic factors [63]. The results from the present study suggest that extrinsic factors induce more genomic mutations than intrinsic factors in lv-iPSCs. The high rate of tumorigenesis of iPSCs in vivo suggests that extrinsic factors strongly contribute to cancer risk and carcinogenesis.

Conclusions

The present study demonstrated a low level of DNA damage repair in iPSCs. Ionizing radiation induced more somatic mutations and short indels in iPSCs than in ESCs or MEFs. Genome stability was higher in iPSCs induced chemically than in iPSCs induced with lentivirus. The high genome instability of lv-iPSCs appears to reflect increased NHEJ and decreased HR pathways of DNA damage repair, and could contribute to the high rate of tumorigenesis in vivo.

Notes

Declarations

Authors’ contributions

YS, QZ and JC conceived this study. MZ and LW contributed to its design. MZ, GL, KA, CY, HL, FD, XH and YL performed experiments, while MZ and GL performed bioinformatic analyses. JC assisted with bioinformatic analysis and interpretation. YS and MZ interpreted the data and wrote the paper with the assistance of the other authors. QZ, DP, and XX provided critical technical assistance and expertise. All authors read and approved the final manuscript.

Acknowledgements

We thank Professor Qi Zhou from the Institute of Zoology of the Chinese Academy of Sciences for generously supplying iPS- and ES-derived mice. We thank Professor Duanqing Pei from the Guangzhou Institutes of Biomedicine and Health of the Chinese Academy of Sciences for providing the lv-iPSC line, and Xin Xie from the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences for providing the ci-iPSC line. We are also grateful to our laboratory colleagues for their assistance with experiments and manuscript preparation.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The raw sequencing data reported in this manuscript are publicly available at the Genome Sequence Archive (http://gsa.big.ac.cn) under Accession Number CRA000695.

All data generated and analyzed are included in the article to support the conclusions.

Consent for publication

Not applicable.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Beijing Institute of Genomics and the School of Life Sciences at the Chinese Academy of Sciences.

Funding

This work was supported by the Precision Medicine Research Program of the Chinese Academy of Sciences (KJZD-EW-L14), Strategic Priority Research Program of the Chinese Academy of Sciences (XDA01040407), National Natural Science Foundation of China (31471395, 91019024, 31540033 and 31100558), National Basic Research Program of China (973 Program, 2012CB518302 and 2013CB911001) and 100 Talents Project.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Key Laboratory of Genomic and Precision Medicine, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, P. R. China
(2)
University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
(3)
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, P. R. China
(4)
The Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, P. R. China
(5)
CAS Key Laboratory of Receptor Research, the National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, P. R. China

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